Structure /Function of Phospholamban in Heart

描述（由申请人提供）：本研究的长期目标是阐明磷蛋白（PLB）抑制心脏肌浆网（SR）中钙泵（SERCA2a亚型）活性的分子和生物物理机制。PLB是心脏SR中的五聚体磷蛋白，由五个相同的单体组成。之前，我们证明了PLB单体负责与SERCA2a结合并抑制它。现在，我们建议定位PLB单体与SERCA2a之间导致酶抑制的结合相互作用位点，并确定分子相互作用如何受到包括Ca浓度、核苷酸和磷酸化在内的关键变容调节剂的调节。重点将放在识别氨基酸的抑制复合体，直接相互作用，利用我们新开发的化学交联方法的优势。在Aim 1中，我们将对PLB进行Cys扫描诱变，以定位其主要结构上与SERCA2a内源性Cys残基交联的不同位点。SERCA2a交联的Cys残基将通过蛋白纯化/肽段测序直接鉴定。在Aim 2中，SERCA2a交联到PLB不同位点的Lys残基将被定位。通过使用交联剂作为分子标尺并结合Aims 1和Aims 2的结果，我们将建立PLB单体与SERCA2a之间形成的结合复合物的精确三维模型。在Aim 3中，将研究Ca浓度、核苷酸和抑制剂thapsigargin对PLB与SERCA2a交联的影响。经检验的假设是，PLB只与SERCA2a的无ca形式（E2）结合，但仅限于与ATP或ADP结合的E2状态。在Aim 4中，我们将确定Ca如何缓解PLB对SERCA2a的抑制。我们假设PLB优先与E2结合，拮抗Ca与SERCA2a的结合，而Ca优先与El结合，拮抗PLB与SERCA2a的结合。将鉴定SERCA2a中负责将PLB与泵解离的ca结合位点，并量化PLB对SERCA2a的ca结合亲和力的影响。在Aim 5中，我们将确定蛋白激酶对PLB的磷酸化如何减轻PLB抑制。经过验证的假设是，PLB的磷酸化直接使其与SERCA2a分离。在这里，我们还将确定PLB的两个磷酸化残基，Ser 16和Thr 17是否直接与SERCA2a相互作用，以帮助抑制酶。PLB是心肌收缩动力学的关键调节因子。通过确定其对钙泵作用的分子机制，将对PLB调节心跳强度产生新的见解，这可能最终导致治疗心力衰竭的新药的设计。

DESCRIPTION (provided by applicant): The long term goal of this research is to elucidate the molecular and biophysical mechanism by which phospholamban (PLB) inhibits the activity of the Ca pump (SERCA2a isoform) in cardiac sarcoplasmic reticulum (SR). PLB is a pentameric phosphoprotein in cardiac SR, which is composed of five identical monomers. Previously, we demonstrated the PLB monomer is responsible for binding to SERCA2a and inhibiting it. Now, we propose to localize the binding-interaction sites between the PLB monomer and SERCA2a that lead to enzyme inhibition, and determine how the molecular interaction is regulated by key allosteric modulators including Ca concentration, nucleotides, and phosphorylation. Emphasis will be placed upon identifying amino acids in the inhibitory complex that interact directly, taking advantage of our newly developed chemical cross-linking method. In Aim 1, we will perform Cys-scanning mutagenesis of PLB to localize distinct sites along its primary structure that cross-link to endogenous Cys residues of SERCA2a. The cross-linked Cys residues of SERCA2a will be directly identified by protein purification/peptide sequencing. In Aim 2, Lys residues of SERCA2a that cross-link to distinct sites of PLB will be localized. By use of crosslinking agents as molecular rulers and combining results from Aims 1 and 2, we will develop an accurate 3-D model of the binding-complex formed between the PLB monomer and SERCA2a. In Aim 3, the effects of Ca concentration, nucleotides, and the inhibitor thapsigargin on cross-linking of PLB to SERCA2a will be investigated. The hypothesis tested is that PLB binds exclusively to the Ca-free form (E2) of SERCA2a, but only that E2 state that has bound ATP or ADP. In Aim 4, we will determine how Ca relieves PLB inhibition of SERCA2a. We hypothesize that PLB binds preferentially to E2, antagonizing Ca binding to SERCA2a, and that Ca binds preferentially to El, antagonizing PLB binding to SERCA2a. The Ca-binding site of SERCA2a responsible for dissociating PLB from the pump will be identified, and the effect of PLB on the Ca-binding affinity of SERCA2a will be quantified. In Aim 5, we will determine how phosphorylation of PLB by protein kinases relieves PLB inhibition. The hypothesis tested is that phosphorylation of PLB directly dissociates it from SERCA2a. Here we will also determine if the two phosphorylated residues of PLB, Ser 16 and Thr 17, interact directly with SERCA2a to aid in enzyme inhibition. PLB is a key regulator of myocardial contractile dynamics. By defining its molecular mechanism of action on the Ca pump, new insights on PLB regulation of the strength of the heartbeat will result, that may ultimately lead to the design of new drugs to treat heart failure.